Ribonucleotide reductase is responsible for regulating a balanced and continued supply of deoxyribonucleotide precursors required for DNA replication. As an approach to understanding the structural and functional basis for regulation of ribonucleotide reduction we have isolated and characterized a membrane complex containing ribonucleotide reductase protein B1, B2 and thioredoxin. This form of ribonucleotide reductase is characterized as a highly active, and physically stable complex. Unlike the solubilized form of reductase which shows dilution inactivation, the specific activity in the membrane fraction is independent of protein concentration. The functional significance and the integrity of this membrane reductase complex is also suggested by the co-purification of several other enzymes involved in deoxynucleotide metabolism, including thymidylate synthetase and DNA polymerase I. Present studies will continue characterization of the various enzyme activities co-purifying with the ribonucleotide reductase-membrane fraction together with studies on any functional relations that exist between these proteins. The enzymatic behavior of the membrane fraction as well as the B1 protein purified as a lipoprotein complex on dATP sepharose. Thioredoxin, the hydrogen donor implicated in ribonucleotide reduction, has been isolated as a phosphoprotein. Phosphate is localized on the thiol of cysteine 32. In vivo 96% of all thioredoxins were phosphorylated. Sequence analysis demonstrated that the phosphorylated amino acid is the same cysteine involved in the hydrogen donor reaction. Physiological studies will investigate the implied role of phosphothioredoxin in phosphate transfer reactions.